Corynebacterium diphtheriae putative tellurite-resistance protein (CDCE8392_0813) contributes to the intracellular survival in human epithelial cells and lethality of Caenorhabditis elegans

Corynebacterium diphtheriae, the aetiologic agent of diphtheria, also represents a global medical challenge because of the existence of invasive strains as causative agents of systemic infections. Although tellurite (TeO3^2-) is toxic to most microorganisms, TeO3^2--resistant bacteria, including C. diphtheriae, exist in nature. The presence of TeO3^2--resistance (Te^R) determinants in pathogenic bacteria might provide selective advantages in the natural environment. In the present study, we investigated the role of the putative Te^R determinant (CDCE8392_813gene) in the virulence attributes of diphtheria bacilli. The disruption of CDCE8392_0813 gene expression in the LDCIC-L1 mutant increased susceptibility to TeO3^2- and reactive oxygen species (hydrogen peroxide), but not to other antimicrobial agents. The LDCIC-L1 mutant also showed a decrease in both the lethality of Caenorhabditis elegans and the survival inside of human epithelial cells compared to wild-type strain. Conversely, the haemagglutinating activity and adherence to and formation of biofilms on different abiotic surfaces were not regulated through the CDCE8392_0813 gene. In conclusion, the CDCE8392_813 gene contributes to the Te^R and pathogenic potential of C. diphtheriae.     

PKC-1 acts with the ERK MAPK signaling pathway to regulate Caenorhabditis elegans mechanosensory response

In most animals, multiple genes encode protein kinase C (PKC) proteins. Pharmacological studies have revealed numerous roles for this protein family, yet the in vivo roles of specific PKC proteins and the functional targets of PKC activation are poorly understood. We find that in Caenorhabditis elegans, two PKC genes, pkc-1 and tpa-1, are required for mechanosensory response; the role of the nPKCε/η ortholog, pkc-1, was examined in detail. pkc-1 function is required for response to nose touch in adult C. elegans and pkc-1 likely acts in the interneurons that regulate locomotion which are direct synaptic targets of mechanosensory neurons. Previous studies have suggested numerous possible targets of pkc-1; our analysis indicates that pkc-1 may act via the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway. We find that ERK/MAPK pathway function is required for mechanosensory response in C. elegans and that at least one component of this pathway, lin-45 Raf, acts in interneurons of the mechanosensory circuit. Genetic analysis indicates that lin-45 and pkc-1 act together to regulate nose touch response. Thus, these results functionally link two conserved signaling pathways in adult C. elegans neurons and define distinct roles for PKC genes in vivo.     

秀丽隐杆线虫先天免疫信号转导途径

秀丽隐杆线虫因其结构简单、易于培养、生命周期短等特点作为一种模式生物已广泛应用于神经系统、衰老机制及细胞程序性死亡的研究。与高等生物不同,秀丽隐杆线虫缺少适应性免疫途径,只有先天免疫途径在抗病原菌、抗氧化应激等方面发挥重要的作用。其体内的胰岛素/胰岛素样生长因子(insulin/ IGF-1)、转化生长因子β(transforming growth factor β,TGF-β)、丝裂原激活的蛋白激酶(mitogen activated protein kinases,MAPK)和细胞程序性死亡(programmed cell death,PCD)4条免疫相关信号转导途径在不同的环境发挥着主要作用。同时,秀丽隐杆线虫的先天免疫系统在进化中有许多保守之处,这为高等生物的免疫机制研究提供了新思路。据此,就有关秀丽隐杆线虫先天免疫信号转导途径的研究进展进行了简述,期望能为人类等高等生物相关联的免疫作用研究提供借鉴和参考。     

骨骼肌特异表达线虫ω-3脂肪酸去饱和酶基因载体的构建与验证

根据猪α-actin基因已知DNA序列设计合成了两个特异性引物,以猪基因组DNA为模板,通过PCR扩增得到α-actin 5'调控序列,然后与线虫ω-3脂肪酸去饱和酶基因cDNA、去除CMV启动子的表达载体pcDNA3.1连接构成肌肉特异性表达载体pcDNA3.1-AF,小鼠股四头肌注射该重组载体,RT-PCR检测证明...     

TAT蛋白转导肽介导的秀丽线虫体内外源蛋白的跨膜转导研究

TAT蛋白转导肽是HIV-1病毒编码的一段富含碱性氨基酸序列的多肽,能够高效介导多种外源生物大分子通过多种膜性结构,如细胞质膜和血脑屏障等。为探索TAT蛋白转导肽介导的秀丽线虫体内外源蛋白跨膜转导作用,以EGFP为报告基因结合常规分子克隆技术构建了原核表达载体pET28b-EGFP和pET28-TAT-EGFP,继而利用诱导剂IPTG(终浓度1mmol/L)诱导表达了靶蛋白并结合荧光显微观察、SDS-PAGE和Western blot等鉴定技术获得表达靶蛋白的大肠杆菌BL21(DE3)细胞,最后将其涂布到含有Kana⁺的LB固体培养基上直接饲喂野生型N2株系线虫,利用荧光显微镜观察绿色荧光信号在线虫体内的分布。结果证明,TAT-EGFP融合蛋白较之于EGFP可高效、可溶性表达,而且通过直接饲喂秀丽线虫表达靶蛋白的大肠杆菌48小时后,TAT-EGFP荧光信号明显分布于线虫肠壁细胞,而EGFP荧光信号则分布在秀丽线虫肠腔,空载体对照组未见任何荧光信号,说明TAT蛋白转导肽能够高效介导外源蛋白在秀丽线虫体内跨膜转导。同时,通过比较空载体对照组与实验组线虫微分干涉图像,未见线虫出现明显的细胞形态变化,说明TAT蛋白转导肽介导的外源蛋白跨膜转导作用是安全的,为在秀丽线虫体内直接研究外源蛋白的功能以及进行蛋白药物的研发提供了重要参考。     

In vivo Laser Axotomy in C. elegans

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron is damaged by injury or disease, it may regenerate. Cell-intrinsic and extrinsic factors influence the ability of a neuron to regenerate and restore function. Recently, the nematode C. elegans has emerged as an excellent model organism to identify genes and signaling pathways that influence the regeneration of neurons^1-6. The main way to initiate neuronal regeneration in C. elegans is laser-mediated cutting, or axotomy. During axotomy, a fluorescently-labeled neuronal process is severed using high-energy pulses. Initially, neuronal regeneration in C. elegans was examined using an amplified femtosecond laser⁵. However, subsequent regeneration studies have shown that a conventional pulsed laser can be used to accurately sever neurons in vivo and elicit a similar regenerative response^1,3,7.We present a protocol for performing in vivo laser axotomy in the worm using a MicroPoint pulsed laser, a turnkey system that is readily available and that has been widely used for targeted cell ablation. We describe aligning the laser, mounting the worms, cutting specific neurons, and assessing subsequent regeneration. The system provides the ability to cut large numbers of neurons in multiple worms during one experiment. Thus, laser axotomy as described herein is an efficient system for initiating and analyzing the process of regeneration.     

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, messages are carried by neurotransmitters released from presynaptic terminals and received by receptors on postsynaptic cells. Our previous research in Caenorhabditis elegans has shown that VSM-1 negatively regulates exocytosis. Additionally, analysis of synapses in vsm-1 mutants showed that animals lacking a fully functional VSM-1 have increased synaptic connectivity. Based on these preliminary findings, we hypothesized that C. elegans VSM-1 may play a crucial role in synaptogenesis. To test this hypothesis, double-labeled microarray analysis was performed, and gene expression profiles were determined. First, total RNA was isolated, reversely transcribed to cDNA, and hybridized to the DNA microarrays. Then, in-silico analysis of fluorescent probe hybridization revealed significant induction of many genes coding for members of the major sperm protein family (MSP) in mutants with enhanced synaptogenesis. MSPs are the major component of sperm in C. elegans and appear to signal nematode oocyte maturation and ovulation . In fruit flies, Chai and colleagues ¹ demonstrated that MSP-like molecules regulate presynaptic bouton number and size at the neuromuscular junction. Moreover, analysis performed by Tsuda and coworkers ² suggested that MSPs may act as ligands for Eph receptors and trigger receptor tyrosine kinase signaling cascades. Lastly, real time PCR analysis corroborated that the gene coding for MSP-32 is induced in vsm-1(ok1468) mutants. Taken together, research performed by our laboratory has shown that vsm-1 mutants have a significant increase in synaptic density, which could be mediated by MSP-32 signaling.     

RNAi mediated gene knockdown and transgenesis by microinjection in the necromenic Nematode Pristionchus pacificus

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression analyses by RNA interference and stable transgenesis remain limited in many species due to the particular anatomy and molecular cellular biology of the organism. For example, outside of the crown group Caenorhabditis that includes Caenorhabditis elegans, stably transmitted transgenic lines in non-Caenorhabditis species have not been reported in this specious phylum (Nematoda), with the exception of Strongyloides stercoralis and Pristionchus pacificus. To facilitate the expanding role of P. pacificus in the study of development, evolution, and behavior, we describe here the current methods to use microinjection for making transgenic animals and gene knock down by RNAi. Like the gonads of C. elegans and most other nematodes, the gonads of P. pacificus is syncitial and capable of incorporating DNA and RNA into the oocytes when delivered by direct microinjection. Unlike C. elegans however, stable transgene inheritance and somatic expression in P. pacificus requires the addition of self genomic DNA digested with endonucleases complementary to the ends of target transgenes and coinjection markers. The addition of carrier genomic DNA is similar to the requirement for transgene expression in Strongyloides stercoralis and in the germ cells of C. elegans. However, it is not clear if the specific requirement for the animals' own genomic DNA is because P. pacificus soma is very efficient at silencing non-complex multi-copy genes or that extrachromosomal arrays in P. pacificus require genomic sequences for proper kinetochore assembly during mitosis. The ventral migration of the two-armed (didelphic) gonads in hermaphrodites further complicates the ability to inject both gonads in individual worms. We also demonstrate the use of microinjection to knockdown a dominant mutant (roller,tu92) by injecting double-stranded RNA (dsRNA) into the gonads to obtain non-rolling F(1) progeny. Unlike C. elegans, but like most other nematodes, P. pacificus PS312 is not receptive to systemic RNAi via feeding and soaking and therefore dsRNA must be administered by microinjection into the syncitial gonads. In this current study, we hope to describe the microinjection process needed to transform a Ppa-egl-4 promoter::GFP fusion reporter and knockdown a dominant roller prl-1 (tu92) mutant in a visually informative protocol.     

The Pseudomonas aeruginosa PA14 type III secretion system is expressed but not essential to virulence in the Caenorhabditis elegans-P. aeruginosa pathogenicity model

The Pseudomonas aeruginosa type III secretion system (TTSS), enabling direct injection of toxins into host cells, has been shown to be crucial to virulence in several models of P. aeruginosa pathogenesis. Using the strain PA14 and its isogenic mutant, PA14exsA, we investigated the role of the TTSS during infection of the nematode Caenorhabditis elegans. Although C. elegans N2 was killed by PA14 in an infection like process over 48 to 72 h the same effect was observed following infection with PA14exsA, implying that a functional TTSS was not essential for virulence. This was despite the TTSS being actively expressed during C. elegans infection as demonstrated by the use of green fluorescent reporter constructs and RT-PCR. However, compared to the wild type PA14, PA14exsA did display a reduced rate of killing of C. elegans strain AU1 which harbours a mutation in the sek-1 gene encoding a MAP kinase involved in nematode innate immunity. A fuller understanding of the mechanism of resistance to type III attack in C. elegans may lead to the identification and development of novel therapeutic targets affording protection to TTSS products in man.     

Structural characterization of phytotoxic terpenoids from Cestrum parqui

Isolation, chemical characterization and phytotoxicity of nine polyhydroxylated terpenes (five C13nor-isoprenoids, two sesquiterpenes, a spirostane and a pseudosapogenin) from Cestrum parqui L'Herr are reported. In this work we completed the phytochemical investigation of the terpenic fraction of the plant and described the structural elucidation of polar isoprenoids using NMR methods. All the configurations of the compounds have been assigned by NOESY experiments. Four new structures have been identified as (3S,5R,6R,7E,9R)-5,6,9-trihydroxy-3-isopropyloxy-7-megastigmene, 5alpha-spirostan-3beta,12beta,15alpha-triol, and 26-O-(3'-isopentanoyl)-beta-d-glucopyranosyl-5alpha-furost-20(22)-ene-3beta,26-diol, and as an unusual tricyclic sesquiterpene. The compounds have been assayed for their phytotoxicity on lettuce at the concentrations ranging between 10(-4) and 10(-7)M. The activities of some compounds were similar to that of the herbicide pendimethalin.